The therapy of viral infection is in its infancy. Bacterial infection is typically treated with agents, such as antibiotics, which take advantage of the differences in metabolism between the infecting organism and its host. However, viruses largely employ the host's own enzymes to effect their replication, and thus leave few opportunities for pharmacological intervention. By employing strong regulatory elements, the virus obtains transcription and translation of its own genes at the expense of host genes.
In mammals, viral infection is combatted naturally by cytotoxic T-lymphocytes, which recognize viral proteins when expressed on the surface of host cells, and lyse the infected cells. Destruction of the infected cell prevents the further replication of virus. Other defenses include the expression of interferon, which inhibits protein synthesis and viral budding, and expression of antibodies, which remove free viral particles from body fluids. However, induction of these natural mechanisms require exposure of the viral proteins to the immune system. Many viruses, for example herpes simplex virus-l (HSV-1), exhibit a dormant or latent phase, during which little or no protein synthesis is conducted. The viral infection is essentially invisible to the immune system during such phases.
Retroviruses carry the infectious form of their genome in the form of a strand of RNA. Upon infection, the RNA genome is reverse-transcribed into DNA, and is typically then integrated into the host's chromosomal DNA at a random site. On occasion, integration occurs at a site which truncates a gene encoding an essential cellular receptor or growth factor, or which places such a gene under control of the strong viral cis-acting regulatory element, which may result in transformation of the cell into a malignant state.
Viruses may also be oncogenic due to the action of their trans-acting regulatory factors on host cell regulatory sequences. In fact, oncogenesis was the characteristic which lead to the discovery of the first known retroviruses to infect humans. HTLV-I and HTLV-II (human T-lymphotropic viruses I and II) were identified in the blood cells of patients suffering from adult T-cell leukemia (ATL), and are believed to induce neoplastic transformation by the action of their trans activating factors on lymphocyte promoter regions. HTLV-I and II preferentially infect human lymphocytes, and on occasion, cause their transformation into malignancy. Since then, two additional retroviruses have been found to infect humans: HIV-I and HIV-II, the etiological agents of AIDS. However, HIV-I and II apparently contribute to cancer only through their immunosuppressive effects.
HIV I and II apparently infect cells which express the CD4 surface protein. This protein is present in abundance on thymocytes and some T-lymphocytes, and to a lesser extent, on some antigen-presenting cells. HIV infection is initially characterized by flu-like symptoms, followed by a long latency period, which may last five to ten years. Upon entering its active phase, HIV infection results in a rapid decline in the population of "helper" T-lymphocytes (TH), which is usually recognized as a decline in the ratio of T4.sup.+ /T8.sup.+ (CD4.sup.+ /CD8.sup.+) T-lymphocytes. The patient typically experiences severe diarrhea, and if the central nervous system is infected, exhibits a form of dementia. The depletion of T.sub.H cells cripples the immune system, and the patient succumbs to an opportunistic infection by, for example P. carinii or cytomegalovirus, or to Karposi's sarcoma. The natural immune system appears wholly incapable of combatting HIV infection, despite the typical presence of apparently neutralizing serum antibody titers during latency.
Current therapy for HIV infection per se is limited mainly to administration of AZT to inhibit viral progression, although M. S. Hirsch, J Infect Dis (1988) 157:427-31 reported synergistic inhibition of HIV by AZT with GM-CSF (granulocyte-monocyte colony stimulating factor) or .alpha. interferon. AZT (3'-azido-3'-deoxythymidine) is representative of a class of dideoxynucleoside (ddN) antiviral agents. These agents rely on the ability of host DNA polymerases to reject a ddN, and the tendency of viral polymerases to accept ddNs and incorporate them into replicating polynucleotides. Upon incorporation, a ddN stops polymerization, as it lacks the 3' hydroxyl group necessary for the next phosphodiester linkage. The ddNs are typically inactive in their administered form, and depend on phosphorylation by host cell enzymes for conversion to the active triphosphate ddNTP.
H. Mitsuya et al, Nature (1987) 325:773-78 disclosed the organization of the HIV genome, and suggested various strategies for development of HIV therapies. Speculated therapies included administration of antisense RNA (as free strands or encoded by an "antivirus") to inhibit viral transcription, administration of glycosylation inhibitors, administration of interferons to inhibit viral budding, and administration of dideoxynucleoside analogues to inhibit viral replication. Dideoxynucleoside analog drugs useful for HIV therapy (e.g., AZT, ddC, etc.) depend on host cell enzymes for activation by phosphorylation. D. Baltimore, Nature (1988) 335:395-96 suggested treating AIDS by removing bone marrow cells from an infected subject, and transfecting hematopoietic cells in the bone marrow extract with DNA or virus encoding an RNA or protein able to interfere with HIV growth. The DNA could encode RNA that might bind to HIV regulatory proteins, antisense RNA, mutant viral polypeptides, or a viral DNA-binding protein lacking its regulator function. The transfected cells would then be re-introduced into the subject, and provided with a selective advantage to insure dissemination.
A. I. Dayton et al, Cell (1986) 44:941-47 disclosed that the HIV tat gene is essential for viral protein synthesis and replication. Dayton suggested that one might inhibit HIV by interference with tat, without otherwise affecting the host cell. M. A. Muesing et al, Cell (1987) 48:691-701 disclosed that the tat protein (rather than tat mRNA alone) is essential for transactivation. Muesing also found that a tat-art fusion (having 114 amino acids fused to the C-terminus of tat by deletion of 7 nucleotides) retained full tat activity. A. D. Frankel et al, Science (1988) 240:70-73 disclosed that tat forms metal-linked dimers in vitro, and suggested that possible treatments for AIDS may involve chelation of metal ions, or competition for tat monomer binding. A. D. Frankel et al, Proc Nat Acad Sci USA (1988) 85:6297-30 disclosed the synthesis of an HIV-1 tat fragment which retains the metal-binding properties of tat. The fragment formed heterodimers with native tat, and can displace tat in homodimers. Frankel suggested using the tat fragment to inhibit tat dimerization, using liposomes to deliver the peptides or a tat-fragment gene. The amino acid sequence reported for tat from one HIV-1 isolate is
______________________________________ Met Glu Pro Val Asp Pro Arg Leu Glu Pro Trp Lys His Pro Gly Ser Gln Pro Lys Thr Ala Cys Thr Asn Cys Tyr Cys Lys Lys Cys Cys Phe His Cys Gln Val Cys Phe Ile Thr Lys Ala Leu Gly Ile Ser Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Gly Ser Gln Thr His Gln Val Ser Leu Ser Lys Gln Pro Thr Ser Gln Ser Arg Gly Asp Pro Thr Gly Pro Lys Glu. ______________________________________
A similar protein is found in HIV-2.
The tar sequence, which acts in cis and is regulated by HIV tat, is found approximately at nucleotides +19 to +82 in the HIV-1 genome. The sequence contains two extended, inverted repeats, and is thus predicted to be able to form stem loop structures (Muessing, supra). HIV-2 exhibits a similar region.
Haseltine, U.S. Pat. No. 4,738,922 disclosed the HTLV I and II LTR promoter regions, and their use with trans-activators (luk) in vectors for amplified expression of heterologous proteins, exemplified by chloramphenicol acetyltransferase (CAT). HTLV-I LTR apparently promotes constitutive expression, which is further amplified in the presence of HTLV-I luk, while HTLV-II LTR apparently requires HTLV-II luk for expression. Haseltine mentioned that viral trans-acting factors can alter the expression of host cell genes as well as viral genes, and Haseltine disclosed the concept of inserting a vector having the HTLV LTR fused to a heterologous gene into a cell having the HTLV genome, which results in the trans-activation of the heterologous gene and the overexpression of its product. Haseltine disclosed the use of the HTLV LTR in the absence of luk to generate empty capsid vaccines, and suggested using the HTLV LTR for expression of antigens, to provide for destruction of the cell by monoclonal or polyclonal antibodies.
E. A. Dzierzak et al, Nature (1988) 331:35-41 disclosed a prototypical gene therapy method in mice. Dzierzak removed bone marrow cells from mice, exposed the mice to lethal radiation, and introduced human .beta.-globin (BG) genes into the removed marrow using a retroviral vector, pSV(X)neo. The BG gene was inserted to read in the opposite direction from proviral transcription, and constructs both with and without the viral LTR enhancer regions were prepared. The mice were then reconstituted with the recombinant bone marrow, containing hematopoietic stem cells. Dzierzak found that human .beta.-globin was expressed in the resulting recombinant mice, and that the expression was found only in cells of erythroid lineage. J-K Yee et al, Proc Nat Acad Sci USA (1987) 84:5197-201 disclosed construction of retroviral vectors encoding HPRT under control of either the metallothionein promoter or the human cytomegalovirus (hCMV) promoter. Yee found that HPRT expression doubled when transcriptional regulatory sequences were deleted from the U3 region of the retroviral LTR.
S.-F. Yu et al, Proc Nat Acad Sci USA (1986) 83:3194-98 disclosed the construction of self-inactivating ("SIN") retroviral gene transfer vectors. SIN vectors are created by deleting the promoter and enhancer sequences from the U3 region of the 3' LTR. A functional U3 region in the 5' LTR permits expression of the recombinant viral genome in appropriate packaging cell lines. However, upon expression of its genomic RNA and reverse transcription into cDNA, the U3 region of the 5' LTR of the original provirus is deleted, and is replaced with the U3 region of the 3' LTR. Thus, when the SIN vector integrates, the non-functional 3' LTR U3 region replaces the functional 5' LTR U3 region, and renders the virus incapable of expressing the full-length genomic transcript. Yu constructed a recombinant virus using the Mo-MuLV LTR regions and packaging (psi) sequence, and inserted a neomycin resistance (Neo) gene under control of either a metallothionein promoter (virus MT-N), an HSV tk promoter (virus TK-N), or a SV40 promoter (virus SV-N) as a selectable marker. Yu also inserted a human c-fos gene under control of a human metallothionein promoter (hMT) into TK-N, and demonstrated inducible transcription of c-fos in NIH 3T3 cells after infection with the recombinant virus.
S. L. Mansour et al, Nature (1988) 336:348-52 disclosed a method for selecting cells after homologous recombination with a linear transfecting vector. A linear vector was prepared having a region homologous to a target gene, a neomycin resistance gene inserted in an exon of the homologous region, and an HSV-tk gene outside the homologous region. Upon specific homologous recombination, the transformed cell displays a phenotype negative for the target region and HSV-tk, and positive for neomycin resistance (homologous recombination into the target site disrupts the target site gene, and fails to incorporate the tk gene). The phenotype distinguishes cells having homologous recombination from non-specific integration, as the latter cells will display a phenotype positive for the target gene, HSV-tk, and neomycin resistance. Neomycin resistance is tested by culturing cells in neomycin, while tk.sup.+ is tested by culturing cells in gancyclovir (which is converted to a toxic product by tk).
R. D. Palmiter et al, Cell (1987) 80:435-43 disclosed the preparation of transgenic mice having a DNA construct encoding the diphtheria A chain under control of the elastase promoter. The elastase promoter is active only in pancreatic acinar cells, and promotes the expression of elastase. The DNA constructs were microinjected into mouse eggs, and the resulting progeny examined. Transgenic mice in which the construct was active failed to develop normal pancreatic tissue.
M. E. Selsted et al, J Clin Invest (1985) 76:1436-39 disclosed the primary amino acid sequence for three related human cytotoxic effector polypeptides, termed human neutrophil antimicrobial peptides (HNPs). The three HNPs have 29-30 amino acid residues and exhibit activity against bacteria, fungi, and herpes simplex virus (T. Gantz et al, J Clin Invest (1985) 76:1427-35).
T. L. Wasmoen et al, J Biol Chem (1988) 263:12559-63 disclosed the primary amino acid sequence of human eosinophil granule major basic protein (MBP). MBP is an effector polypeptide having 117 amino acid residues, a pI of 10.9, and exhibiting cytotoxic activity against mammalian cells and parasites.
M. A. Adam et al, J Virol (1988) 62:3802-06 disclosed retroviral vectors which exhibit efficient RNA packaging, having a psi+sequence. R. D. Cone et al, Mol Cell Biol (1987) 7:887-97 disclosed the construction of retroviral vectors (pSVX) including a human .beta.-globin gene, and the use of the recombinant vectors to obtain human .beta.-globin expression in a murine erythroleukemia cell line. A. D. Miller et al, Mol Cell Biol (1986) 6:2895-902 disclosed cell lines useful for packaging replication-defective retroviral vectors.
Guild et al, J Virol (1988) 62:3795-801 disclosed retroviral vectors using the Mo-MuLV LTRs and psi (packaging) sequence, useful for transfer of entire genes into mammalian cells. Guild employed .beta.-actin and histone promoters (which are active in essentially all cells) to obtain transcription of neo.sup.r (as a selectable marker). Expression of the neo.sup.r was demonstrated in vivo, after virus-infected bone marrow cells were used to reconstitute lethally-irradiated mice.
A. D. Friedman et al, Nature (1988) 335:452-54 disclosed transfection of tk.sup.- mouse cells with plasmids expressing HSV-1 tk, and HSV-1 VP16. VP16 acts as a trans activator for transcription of the immediate early genes in herpes simplex virus (HSV-1). On the VP16 plasmid, the promoter was replaced with the Mo-MSV promoter, and the carboxy terminal of the VP16 was deleted. The resulting plasmid codes for a mutant VP16 protein which competes with wild type VP16 for DNA binding. Transfected cells exhibited resistance to HSV-1 replication upon later infection. Friedman suggested that one might induce resistance to HIV by transfecting with a dominant mutant of the HIV trans-activator protein (tat).
J. Sodroski et al, Science (1985) 229:74-77 disclosed the location of the HIV tat gene. J. Sodroski et al, Science (1985) 227:171-73 disclosed construction of a plasmid having CAT under control of the HIV LTR. The HIV LTR contains the transactivating region (tar) gene which is induced by tat. Sodroski discovered that transactivating factors would induce transcription of genes under control of the HIV LTR. B. M. Peterlin et al, Proc Nat Acad Sci USA (1986) 83:9734-38 disclosed plasmids having the HIV tar gene, and the effects of orientation and position of tar on transactivation by tat. Peterlin found that tar functions best when downstream from a promoter and an enhancer. Activation with tat increased transcription to RNA. G. J. Nabel et al, Science (1988) 239:1299-302 disclosed that a HIV tat-III-CAT fusion plasmid could be activated by HSV and adenovirus trans-acting factors.
B. K. Felber et al, Science (1988) 239:184-87 disclosed an assay for HIV, using CD4-expressing cell lines transfected with the chloramphenicol acetyltransferase (CAT) gene under control of the HIV LTR. Upon infection with HIV, the transfected cell lines expressed CAT in proportion to the amount of virus present. Felber suggested using the assay as a means for screening possible anti-HIV drugs for their ability to inhibit viral growth, and for identifying anti-tat drugs.